CN107708673B - Process for producing polymer microsphere - Google Patents

Abstract

The invention discloses a microsphere production process, which consists of three unit operations: 1) generating microspheres; 2) quality control of the microspheres; 3) and (4) carrying out aftertreatment on the microspheres. First unit operation, or unit operation 1), includes four basic functions: forcing the microsphere-forming material through a porous membrane to form embryonic microspheres; promoting the embryo microspheres to fall off from the porous membrane; solidifying the embryo microspheres; the cured microspheres are collected and output. The quality control unit operations include the discovery and discharge of oversized microspheres. The post-processing unit operation includes two basic functions: to smooth the surface of the microspheres and to reduce the organic solvent residue in the microsphere matrix.

Description

Process for producing polymer microsphere

Related references and patent applications

This application claims priority from us priority application #62/118, 465 filed on day 20/2/2015 and us priority application #62/185, 623 filed on day 28/6/2015. The contents of both of which are incorporated herein by reference.

Technical Field

The present invention describes a process for preparing polymeric microspheres to achieve programmable uniform particle size, improved encapsulation of water-soluble drugs, and reduced unit operations. The prepared microsphere can effectively encapsulate bioactive substances (including therapeutic substances), maintain the natural conformation of the bioactive substances, and realize controlled release or sustained release on human bodies or animals.

Background

Polymeric microspheres have been successfully used for controlled and sustained release of active substances, including therapeutic substances such as chemical drugs or therapeutic polypeptides. Such dosage forms have also been used in the development of controlled or sustained release protein delivery techniques. The ability of the polymeric microspheres to control or sustain the release of the active or therapeutic substance greatly improves the compliance of patients requiring frequent injections for long periods of time or even for life. By virtue of the controlled or sustained release function, the in vivo concentration of the generalized drug (drug and vaccine) can be well maintained within the therapeutic window (a state such that the in vivo level of the drug/generalized drug is above the minimum effective concentration, but below the minimum toxic concentration). The frequency of the undesired injections will thus be considerably reduced.

The above advantages are accompanied by some disadvantages. One of the major challenges in the preparation of protein microspheres is the sterilization process thereof. The microspheres for sustained release administration of biological substances generally have a diameter in the range of 10-100 microns, and therefore cannot be sterilized by a membrane filtration method with a pore size of 0.2 microns commonly used for protein drugs. Radiation exposure and heat sterilization are also undesirable, as proteins or other therapeutic substances are denatured or degraded under harsh conditions due to their fragility. The only viable sterilization method for controlled or sustained release microspheres is to prepare such formulations under aseptic conditions, i.e., to place the entire preparation process in an aseptic environment. The microspheres produced by the existing method have different particle sizes and need additional operations such as pre-freeze drying, sieving, powder filling and the like. The additional screening, powder filling and other unit operations make the preparation isolated in a sterile environment without exposing to the surrounding environment. It is also important that the screening of microspheres of unwanted particle size reduces the yield of production, whereas powder filling requires complex equipment. In addition to size variation, agitation, a unit operation that prevents microsphere adhesion in current manufacturing processes, can cause leakage of the encapsulated material. Shear tension caused by agitation may break the newly formed microspheres, exposing the encapsulated material to the continuous phase and to the water-oil (organic) interface, which is a known factor in protein denaturation. In addition, too small a microsphere causes a burst release, while too large a microsphere blocks the needle unless a large, obnoxious needle is used.

Although sustained release microspheres are the only formulations that can actually last longer than two weeks in a single injection to date, their use is limited to a few drugs due to the difficulty of preparation. Clearly, a manufacturing process that ensures a uniform and programmable particle size, enabling fluid bottling, would greatly improve the aseptic production of microspheres.

Brief description of the invention

Disclosed is a method/process for preparing microspheres, which can achieve a designable uniform particle size, an encapsulation efficiency of water-soluble components of 90% or more, a smooth surface minimizing burst release, and reduction in the number of unit operation steps and ease of operation. This process invention comprises three operating units: forming and curing uniform microspheres; screening out oversize particles; and post-processing after molding, including smoothing of the microsphere surface. Each operation unit has self-consistent process design, so that the operation is simple and convenient, and various factors are easy to control.

The first unit of operation, the forming and curing of microspheres, comprises three basic steps: 1) pressing the particle-forming material through a porous plate and exfoliating the material in the form of embryonic (soft) microspheres in a microsphere receiving/carrying medium; 2) solidifying the embryonic microspheres by extracting the solvent of the microsphere-forming material; 3) the solidified microspheres are collected so that they can be transported out with as little carrier medium as possible. A multi-well plate may also be called a multi-well barrier or a porous membrane, which has a desired pore size, is tubular or otherwise shaped. An SPG membrane manufactured by a company called "SPG technology" is an example of a tubular porous membrane. The microsphere-forming material is typically dissolved in a solvent and can thus be extruded through the pores of the membrane to form embryonic microspheres. The embryonic microspheres emerging from the porous membrane surface must be removed in time into the receiving/carrying fluid to avoid mutual contact and fusion at the membrane surface. In view of this, some shear force or vibration needs to be applied to help the embryo microspheres fall into the carrier fluid.

The fallen soft embryonic microspheres must be cured before the next unit operation, and the curing process must avoid collision, breakage, and fusion of the embryonic microspheres. To accomplish this, the receiving or carrier liquid must be immiscible with the microsphere-forming material or solution thereof, yet capable of extracting the solvent that dissolves the microsphere-forming material. Methylene chloride and water are a good couple of examples and represent the solvent and microsphere receiver solution in which the microspheres are dissolved. The former can dissolve many biodegradable polymers, while the latter is immiscible with the former but has a certain solubility for it. To avoid contact and fusion, it is desirable to drive the soft embryonic microspheres parallel to the collector during solvent extraction or evaporation (i.e., solidification). There may be two ways to achieve this: allowing the receiving fluid to flow, thereby carrying the embryonic microspheres away; or allowing the embryonic microspheres to settle along the solvent extraction path under gravity to a collector. The latter method appears more straightforward.

To improve the efficiency of the post-treatment, the solidified microspheres should be transferred to a post-treatment vessel while minimizing the volume of receiver fluid transferred therewith. Therefore, the solidified microspheres should be enriched as much as possible prior to transfer to minimize the receiving fluid. However, the enriched microspheres should not be packed too tightly, but should remain sufficiently fluid to be mobile for flow transfer to the next unit operation. The microsphere collector should be capable of enriching the microspheres and allowing them to flow out under the drive of the carrier fluid. The flow rate of the carrier liquid should be low enough to limit the amount of liquid, while its linear velocity should be high enough to allow the microspheres to have sufficient kinetic energy to move upward into the aftertreatment vessel.

At the operation level, the three processes, the formation and the release of the microspheres, the solidification (hardening) of the microspheres, and the collection and the transfer of the microspheres can be realized by one-step operation. Such a simple operation simultaneously provides better microsphere quality, such as uniform particle size, encapsulation efficiency of more than 90%, and improved protein stability due to avoidance of contact with water-oil interfaces.

Between the unit operations associated with microsphere formation and the post-treatment unit operations is a quality control unit that is used to discover and screen out unexpectedly formed, oversized microspheres. Although the few too small or too large particles produced during microsphere generation do not affect the long-lasting sustained release profile of the encapsulated substance, too large microspheres can clog injection needles, necessitating the use of larger needles that are undesirable. To prevent oversized microspheres from entering the production line, a suitable mesh screen is selected and placed in a pipeline connecting the two unit operations. In order to avoid the blockage of the material flow, a three-way valve is arranged in front of the screen, so that the intercepted overlarge microspheres can be discharged out of the production line. The three positions of the three-way valve correspond to three functions, the liquid carrying the microspheres is allowed to flow to the post-treatment container (through the screen), the intercepted overlarge microspheres are discharged, and the microsphere forming and curing cavity is exhausted.

Cleaning is another benefit of the simplified production process of this microsphere. The operation is designed to accomplish a number of tasks including surfactant and salt scavenging, smoothing the surface of the microspheres to reduce burst effect, and removing organic solvent residues locked in the microspheres. To accomplish these goals, the cured microspheres are transferred to a container, rinsed with water, or the surface of the polymeric microspheres is partially swollen with a solvent. The partial swelling is sufficient to lower the phase inversion temperature of the polymer constituting the matrix of the microspheres and to render the surface of the microspheres smooth by mild heat treatment, e.g. below 40 ℃. Such warm heating should be of a degree that does not cause denaturation of the bioactive substance supported in the microspheres, while being sufficient to accelerate diffusion of the remaining organic solvent from the middle to the surface of the microsphere matrix.

To accomplish this, the cleaning vessel must have solvent/water introduction and removal, gentle agitation, and heating. To avoid localized overheating, the heating element should contact the cleaning vessel from an external surface, such as a heating jacket commonly used in chemical laboratories. The release of the cleaning solution must also be light to avoid disturbing the microspheres or exposing them to air. The discharge must also avoid the expulsion of microspheres as well. The water/solvent discharge should then take place from above the cleaning liquid. For example, a floating drain is designed to drain the wash solution from above the sample. The floating discharge port should be a screen to prevent the microspheres from being sucked in. Nevertheless, the floating outlet must have a sufficiently large inlet area to reduce the linear velocity of the discharged liquid so that it does not interfere with the settling of the microspheres. This floating discharge opening must also define its active position to avoid collision with the paddle or the vessel wall. The stirring must be light.

As an important part of quality control, excessively large microspheres should be found and removed from the production line to ensure that smaller needles can be used without clogging concerns. The best opportunity to achieve this is to transfer the cured microspheres to the moment of washing and post-treatment. Thus, a screen and a three-way valve may be placed between the microsphere receiver below the curing column and the wash/post-treatment vessel.

To summarize, the process of the present invention allows the preparation of uniform particle size designable microspheres by two actual unit operations, namely the microsphere formation-solidification-collection process and the microsphere smoothing-solvent removal-washing process. The simplified production process is environment-friendly and safe, and the product quality is greatly improved.

The apparatus or device necessary to carry out the microsphere preparation process proposed by the present invention comprises a porous membrane (e.g., a tubular SPG membrane) with programmable pore size, a generator of shear or vibration to break the formed embryonic microspheres off the porous membrane, a column or tube to allow sedimentation or flow of the embryonic microspheres along the path of solvent extraction, and a collector to enrich the solidified microspheres at their bottom for washing (see FIG. 1). The apparatus or installation should also include a channel for delivering the hardened/solidified microspheres to the cleaning/washing vessel. This channel can be arranged at the bottom of the collector to discharge the microspheres, or in the middle of the collector to suck the microspheres out by the pressure difference of the microsphere receiving fluid itself. In the latter case, the passage or conduit should have a flared or funnel-shaped inlet to direct the entry of the microspheres.

Detailed description of the figures

The drawings herein are for the purpose of assisting the reader in better understanding the present invention and are not intended to limit the scope of the present invention.

FIG. 1 is a diagram of an apparatus; the microsphere formation and curing unit comprises a porous membrane (e.g., a tubular SPG membrane) with a designed pore size, a shear generator or oscillation generator for causing the formed microspheres to fall off the membrane surface; the solvent in the embryo microspheres is extracted in the channels of the column or the tube for guiding the microsphere sedimentation or the tube for guiding the microsphere flowing; a collector between which the hardened/cured microspheres are enriched; and a pipeline with a bell mouth for discharging the microspheres. The solvent extraction channels may be vertical or otherwise oriented. A suction pipe for discharging the microspheres may be placed below or inside the collector. The microsphere collector may take the shape of a circle, a tube, or a funnel. The shear force generator or oscillation generator should produce relative motion between the membrane and the carrier liquid by flowing, rotating, shaking, or vibrating. The connection between the solvent extraction channel and the microsphere collector may be of different forms and sizes, as long as it allows passage of the microspheres. The diameter of the solvent extraction channel and the volume of the microsphere collector can meet the requirements of pilot plant preparation and large-scale production.

FIG. 2 shows the design of an annular nozzle for generating shear forces by agitation along the surface of the porous membrane shown in FIG. 1. This nozzle is fixed to the same tube as the porous membrane. The nozzle may be a straight tube for external pressure type porous membrane arrangement.

FIG. 3. design of microsphere generation unit with oscillator, containing a container loaded with microsphere generation material, a tube connecting it to the SPG membrane, and an SPG membrane extruding embryonic microspheres. The scenario is that an oscillator is placed on the container or connecting tube to shake the embryonic microspheres (i.e., the formed beads) off the surface of the membrane shown in FIG. 1.

FIG. 4 shows a design of a quality control unit capable of discriminating and discharging excessively large microspheres, which includes a screen and a three-way valve.

FIG. 5. design of a post-treatment unit for smoothing the surface of microspheres and cleaning hardened microspheres, comprising a microsphere cleaning vessel, a paddle, a floating drain, a temperature controlled heating system, and a gate valve to unload treated microspheres.

FIG. 6 is an electron microscope image of exenatide loaded microspheres prepared using the process of the invention and the apparatus depicted in FIG. 1. The particle size is in the range of 40-60 μm.

Fig. 7 is a graph of plasma concentration of the once-a-month long-acting microspheres injected into monkeys prepared using the process of the present invention and the apparatus depicted in fig. 1. Since the release profile is nearly perfect, the monthly dose (converted from monkey to human) to achieve the target plasma concentration (300pg/ml) is only 25% of the weekly dose of exenatide microspheres, Bydureon, sold on the market.

Figure 8 electron and light microscope images of Erythropoietin (EPO) -loaded microspheres prepared using the process of the present invention and the apparatus described in figure 1. Both of them demonstrated that the prepared microspheres had uniform particle size.

FIG. 9 is a cumulative release profile of EPO-loaded microspheres prepared using the process of the present invention and the apparatus depicted in FIG. 1.

Figure 10. loss of EPO specific activity following injection of each formulation into monkeys.

Detailed description of the invention

Challenges in producing microspheres, particularly for therapeutic injection, include: difficulty in sterilization, variation in particle size, low encapsulation efficiency, and initial burst. Each challenge is present alone, and is a cause of other problems. They cannot be solved independently in turn. As a practical matter, multiple problems must be solved simultaneously and easily. The present invention provides a greatly simplified process and related device design to produce microspheres of uniform particle size, high efficiency in encapsulation of target components, improved release kinetics, and retained protein conformation.

Integral design of the process

The microsphere production process with simple operation comprises two unit operations, namely a microsphere forming-solidifying-collecting unit and a microsphere smoothing-residual solvent removing-cleaning unit. Physically, this process includes three physical spaces from top to bottom (if placed upright): a sample introduction space, a sample heating-cleaning space, and a sample refrigerating space.

Control of microsphere particle size

The preparation of microspheres with uniform particle size is realized by extruding a microsphere-forming material, generally a drug-loaded polymer solution, through a perforated plate (or rampart or membrane) with a well-designed pore size into a receiving liquid. The resulting embryonic microspheres (beads of drug-loaded polymer solution) must be driven off the surface of the membrane by shear force or vibration at a sufficiently fast rate to avoid fusing with each other prior to shedding. In order to make the embryo microsphere fall off the surface of the porous membrane in time, a flow generator is used to generate light water flow to take away the drop beads, or a vibrator is used to shake the container to make the drop beads fall off. Figures 1,2 and 3 illustrate how the flow generator and oscillator are designed and placed in the system. The design in the figures helps one skilled in the art to easily understand the engineering principle. They should not be used to limit the invention to a specific design, as any form of flow generator or oscillator can achieve the purpose.

The flow generator may be a stirrer creating a circling around the porous membrane, or a nozzle spraying a stream of water. The stirring speed is 50-500 rpm, the flow rate of the nozzle is adjusted between 100-5000 mL/min (the best flow rate is 200-1000 mL/min), the oscillator is fixed on the tube for placing the porous membrane, the frequency is adjusted between 100-500/min, and the best flow rate is 200-400/min. .

The drug molecule may be a chemical molecule or a biological molecule. Biomolecules include proteins, polypeptides, nucleic acids such as siRNA or genes. The biomolecules may be carried in the solution of the polymer from which the microspheres are formed, in the form of solution beads or solid particles.

Solidification of embryonic microspheres

Microspheres that have just been formed on a porous membrane, termed embryonic microspheres, must be received by a continuous phase, suspended and solidified therein. This continuous phase must be immiscible with the particle (microsphere) generating material so that the embryonic microspheres retain their shape. On the basis of immiscibility, the continuous phase must have a certain solubility in the solvent in which the microsphere-forming material is dissolved. The microspheres produced by the porous membrane are solidified by extracting the solvent.

In order to maintain the designed uniform size, the embryo microspheres that are still soft are not allowed to collide with each other and fuse together or are broken by the shearing force of stirring during the solidification process. Current unit operations for microsphere consolidation include agitation of the continuous receiving phase to reduce fusion of the embryonic microspheres. Since these movements of the embryonic microspheres in turn increase the collision between the beads, stirring does not prevent fusion, but rather breaks the fused large microspheres because they are less resistant to tension. In other words, agitation regulates the particle size through a balance between disruption and fusion of the embryonic microspheres.

In the present invention, collision of the embryonic microspheres is avoided by "parallel motion" of the just-formed beads along the elongated channels, and the solvent of the polymer is gradually extracted by the receiving phase during this process. The solvent extraction channels may be placed vertically or in other orientations in which the embryonic microspheres settle under gravity or flow under drive. Sedimentation of the embryonic microspheres in vertical channels to achieve parallel motion is the simplest arrangement.

Such stir-free microsphere curing has the additional advantage that leakage of the encapsulated water-soluble component of the microsphere is avoided or reduced. Because there is no shear force in this process, the embryonic microspheres do not break, and the chance of exposure of the encapsulated water-soluble ingredient to the continuous phase is greatly reduced. More importantly, when the encapsulated ingredient is a fragile protein, avoiding exposure to the continuous/receptive phase of the aqueous medium means preventing these macromolecules from contacting the interface of water-oil (water and polymer solution immiscible therewith), a known factor in protein denaturation. The surfactant, salt, or other adjuvant may be added to the container as with the stirring process.

Temperature of the receiving phase

The path of the microspheres as they settle or otherwise move can be shortened by adjusting the temperature of the continuous receiving phase to increase the solubility of the solvent of the microsphere forming material. For example, the solubility of methylene chloride, a commonly used solvent for the preparation of polymeric microspheres, in water, increases from 2% to 5% when the temperature is decreased from 25 ℃ to 2 ℃. Increasing the aqueous solubility of the solvent facilitates solvent extraction.

Collection and output of microspheres

The microspheres hardened by extraction of the solvent in the long channels settle at the bottom of the vessel and, if vertically positioned, should be concentrated as much as possible to reduce the amount of continuous/receiving phase carried over on the output. Minimizing the volume of the continuous phase is important to enhance cleaning of the microspheres, removal of residual organic solvent from the microsphere matrix, and cleaning of the auxiliary materials in the continuous phase. The design of the vessel can help to enrich the microspheres. FIG. 1 shows, but is not limited to, that the design of the bottom of the container allows the hardened microspheres to be enriched and concentrated. The steeped design at the center of the container allows the microspheres to slide down for enrichment. The steepened portion may be tubular, circular, or funnel-shaped. The bottom most portion of the container should be flat to minimize dead volume when delivering solidified microspheres by aspiration with a pipette.

Transfer of microspheres to a post-treatment Process

Enriched microspheres can be exported by different methods. FIG. 1 shows, but is not limited to, two output designs, either bottom discharge, or suction with a suction tube. The suction pipe must have a trumpet or funnel shaped inlet. A different approach is to pump the hardened microspheres tangentially to the bottom of the vessel along with the continuous phase (not shown in figure 1). The key point in the set-up is that the gap between the trumpet or funnel shaped inlet and the bottom of the vessel must be small enough to produce sufficient linear velocity of the receiving liquid at a reasonable flow rate to carry the microspheres away. This gap is optimized between 1 and 20mm, preferably between 3 and 10mm, but depending on the throughput of production.

On-line quality control

Although the microsphere production process of the present invention can produce microspheres of uniform particle size, it is critical to ensure removal of the oversized microspheres through quality control. One elusive disadvantage of long acting sustained release microsphere formulations is the use of a relatively thick needle. It is always desirable to be able to use as small a needle as possible. However, even a few or even an oversized microsphere can block the needle during injection. From a production efficiency perspective, it is desirable to remove the oversized microspheres through an on-line quality control setting. In the present invention, such a quality control unit is placed between two unit operations, namely 1) the microsphere formation, solidification, collection unit and 2) the microsphere smoothing, desolvation, washing unit. This control unit serves two functions, selectively blocking the oversized microspheres and discharging the oversized microspheres from the production line. The former is arranged on a pipeline connecting the microsphere collector and the post-treatment container by a screen; and the latter places a three-way valve against the screen. This three-way valve connects three components, a) a microsphere collector; B) an encapsulated aftertreatment vessel; C) and discharging the container. When the gap between A and B is opened, the production of the microspheres is carried out; when the channel from A to C is opened, the materials in the microsphere curing column and the microsphere collector are discharged; when the channel from B to C is opened, the oversize microspheres blocked by the screen are discharged. A schematic diagram of the quality control unit is shown in fig. 4. For efficiency of discharge and unloading, the three-way valve should be placed lower, but above the refrigerated space.

Scale of preparation

The scale of preparation according to the above-described microencapsulation process can easily be adjusted by varying the volume of the receiving (continuous) phase, i.e. the size of the porous membrane, the diameter of the settling legs, and the size of the microsphere collector. Thus, the preparation scale can be from several hundred milligrams to several kilograms.

Continuous production

Continuous production requires simultaneous and continuous addition and discharge (or aspiration from the middle) of the microsphere-receiving phase. It would be helpful to have two linked valves to control the addition and discharge (or aspiration). The process of adding and draining (or aspirating) the receiving solution must be slow so as not to affect the settling or flow of the microspheres.

Preparation of microspheres supporting solid particles

Some therapeutic substances, such as proteins, are protected prior to encapsulation in microspheres, which often employ hydrophobic polymers. In such cases, it is common practice to pre-formulate the sensitive drug as microparticles so that they are encapsulated in solid form within the microspheres. This encapsulation process is known as the "solid-in-oil-in-water" (S/O/W) method. The present invention is also applicable to S/O/W processes. The modification required to the encapsulation process of the present invention when used in the S/O/W process is to suspend the particles of the pre-formulation in a polymer solution that forms the microsphere matrix.

Since solid particles may settle at the bottom of the container of polymer solution, it is necessary to subject it to constant stirring or shaking. It is convenient to apply a magnetic field around the container of polymer solution and place a magnetic stirring inside the solution. One embodiment of the present invention is to wind a coil around the outside of the vessel, applying a power source. Compressed air (or other gas such as nitrogen) may also be introduced into the polymer solution in the container to suspend the protein-loaded particles and thereby extrude them out of the porous membrane. The operations of hardening and collecting the protein particle-loaded embryonic microspheres are the same as described above.

Annealing treatment of cured microspheres

To reduce the burst effect of the encapsulated ingredient from the hardened microspheres, the microspheres are preferably smoothed by annealing to remove the solvent-extracted pores. The annealing process may be incorporated into the fabrication process of the present invention.

For polymeric microspheres, the annealing process involves a phase change or partial phase change process of the polymeric material from a glassy state to a colloidal state. The polymeric microspheres will be heated to or above their phase transition temperature, closing the pores at the surface of the microspheres. While the temperature of annealing is affected by the medium in which the microspheres are suspended. For example, when polylactic-co-glycolic acid (PLGA) is used to prepare microspheres, the annealing temperature may be reduced if suspended in an aqueous solution of polyethylene glycol (PEG). The concentration and molecular weight of PEG can be adjusted to reach the designed annealing temperature. For example, with nearly 100% PEG-400, the microsphere surface can be smooth at room temperature. If the concentration of PGE-400 is reduced to 80%, the phase transition temperature of PLGA will rise to 35 ℃. Besides PEG, other agents that are soluble in water and also have certain hydrophobicity can be used to lower the annealing temperature of PLGA microspheres.

Cleaning of microspheres

Prior to filling or prefilling the syringe, the prepared microspheres may need to be cleaned to remove unwanted or later unwanted components introduced during the manufacturing process. This washing/cleaning process involves repeated gentle agitation and aging treatment in fresh water, and replacement of the cleaning solution. The efficient design is to complete the cleaning process and the post-treatment process (also called aging process) at the same time. Therefore, in the vertical placement situation, the microspheres should be separated from the cleaning solution by slow sedimentation so as to avoid the damage of the microspheres. The discharge of the washing liquid is preferably carried out from above the liquid surface in order not to stir up the settled microspheres again. To this end, we have used a floating drain to pump the water out. The inlet to the pump outlet should be large enough to reduce the linear velocity of the discharge while also covering the screen to avoid pumping the microspheres out.

Filling of the final product

Another advantage of the present invention is that the microsphere pharmaceutical product can be fluid bottled. In the existing production process, because the microspheres have different particle sizes, the preparation needs to be dried into powder and then sieved, and oversize or undersize particles are removed. The large microspheres block the needle and the small particles cause burst release. The preparation process can produce microspheres with uniform size, and unit operations such as sieving and powder filling which are not beneficial to aseptic production can be avoided. The collected microspheres can be annealed (according to requirements), and after cleaning, auxiliary materials (such as carboxymethyl cellulose) for regulating and controlling the viscosity of the solution are added, and fluid is filled into bottles, so that unit operation is easier to realize. Fluid filling can simplify the mixing and filling process of powder filling.

Device for measuring the position of a moving object

The device capable of simply and quickly realizing the microsphere preparation process with high quality comprises three basic parts: a microsphere forming and curing unit, a particle size control unit and a post-processing unit. The first section includes a porous membrane through which microsphere-forming material passes to form embryonic microspheres, a channel for extracting solvent to harden the embryonic microspheres, and a collector for enriching the hardened microspheres. The collector may be connected to a drain at the bottom or may house a straw inside. The internally arranged suction pipe is provided with a trumpet-shaped or funnel-shaped inlet. The entire system should also have a reservoir for the final formulation to mix the necessary additives before bottling or plating the product.

Examples

The examples set forth below are similar formulations that we are studying in order to aid the reader in understanding the invention. These examples should not be used to limit the rights of the invention.

EXAMPLE 1 formulation of Exenatide microspheres

First, PLGA/PLA, which is a polymer for forming microspheres, is dissolved in dichloromethane, and exenatide is dissolved in DMSO. The two solutions were then mixed and added to a sample container connected to a tubular porous membrane. Pressurized air or nitrogen (or other gas) is introduced into the vessel and the mixed solution is extruded through the porous membrane into a receiving phase containing polyvinyl alcohol (PVA) and NaCl. The receiving phase is loaded in a column of 1600-1800 lengths, which is connected to a bottle of enriched microspheres. The embryonic microspheres extruded from the porous membrane settle down along the column into the bottle from top to bottom under the action of gravity and harden in about 30-40 seconds of settling time. The hardened microspheres are output to another container for cleaning through a built-in bell mouth suction pipe under the action of the water pressure of the long column. After the microspheres washed with water are proved to have uniform particle size by electron microscope imaging (figure 2), the microspheres are freeze-dried and stored for later use. The particle size is in the range of about 40 to 50 μm.

For some applications, we add Mg (OH) to the polymer solution carrying exenatide₂Or MgCO₃Fine powder, and then extruding the porous film to form the porous film. To improve the release kinetics, the hardened microspheres are annealed by heating to their phase transition temperature (Tg). If drug stability is a concern, the Tg of the polymer can be adjusted (lowered) by adding PEG to the annealed media.

To verify the release kinetics, the above microspheres were injected subcutaneously into normal monkeys, and blood was taken at the designed time to test the exenatide content in the blood. The results are shown in fig. 4, where the microsphere formulation maintains a relatively constant blood level for up to one month with a single needle injection.

Example 2 erythropoietin microsphere formulation

EPO-loaded polysaccharide microparticles obtained via an aqueous-aqueous emulsion or a freeze phase separation pre-formulation were suspended in a PLGA/PLA solution as in example 1. The resulting suspension was added to a container attached to a porous membrane (SPG membrane) and forced through the porous membrane with pressurized nitrogen into the receiving phase as in example 1. All subsequent steps were the same as in example 1. The morphology of the microspheres was confirmed by electron microscopy and light microscopy to be uniform in particle size (see FIG. 5). The particle size is in the range of 70-80 μm.

The release kinetics of EPO and the protective effect of the formulation process were verified by in vitro release experiments and monkey antibody responses. As shown in fig. 6, in vitro experiments showed nearly linear EPO release. FIG. 7 shows a comparison of the antibody responses of EPO microspheres prepared by the method of the present invention and by the double emulsion method reported in the literature. It is clear that the antibody response of the microspheres prepared by the method of the present invention is comparable to the control group such as physiological saline and EPO solution preparations.

Claims (18)

1. A method for preparing microspheres with uniform particle size and high encapsulation efficiency comprises the following steps:

a) extruding the solution of the microsphere-generating material through a porous membrane placed in a receiving solution to generate embryonic microspheres;

b) applying shear force or vibration to the porous membrane to drive the embryonic microspheres generated in step a) away from the surface of the porous membrane;

c) allowing the embryonic microspheres that have been detached in step b) to move under flow or gravity along a channel filled with a receiving liquid to extract the solvent of the microsphere-forming material, thereby hardening the microspheres;

d) collecting the microspheres hardened in step c), wherein

The shear force is generated by the flow or agitation of a receiving liquid in which a porous membrane is placed, or the vibration is generated by shaking or hitting the porous membrane or a fixed member thereof.

2. The method of claim 1, wherein the porous membrane has a defined pore size and can be in the shape of a tube, a circle, or a flat sheet.

3. The method of claim 1 wherein the microsphere receiver is water-based and has the ability to extract the solvent of the microsphere formation material.

4. The method of claim 1, wherein the solvent in which the microsphere-forming material is dissolved is immiscible with water.

5. The method of claim 1, wherein the solidified microspheres are collected at the end of the microsphere receiver fluid channel.

6. The shear force generating member of claim 1, which generates a straight flow through the nozzle and the pipe wall or generates a flow around the pipe wall.

7. The oscillation generator of claim 1, wherein the frequency is adjustable between 100 and 600 times per minute, preferably between 200 and 400 times per minute.

8. The method of claim 1, wherein the channel for receiving the microsphere receiving fluid is positioned vertically, horizontally, or in between.

9. The method of claim 1, wherein the polymer solution is loaded with a biologically active substance.

10. The method of claim 9, wherein the biologically active substance is suspended in particulate form in the polymer solution by stirring or shaking.

11. The method of claim 1 wherein the solidified microspheres are collected and sent to a microsphere formation post-treatment and cleaning vessel.

12. The method of claim 11, wherein a quality control unit is positioned in the microsphere delivery channel to discover and discharge oversized microspheres.

13. The method of claim 12, wherein the quality control unit comprises a screen to prevent passage of the oversized microspheres and a three-way valve to allow the oversized microspheres to exit the production line. .

14. The post-treatment of claim 11 comprising an annealing treatment to smooth the surface of the cured microspheres or to reduce solvent residue.

15. The method of aftertreatment of claim 11, wherein the vessel is provided with an inlet floating above for discharging the aftertreatment or cleaning fluid to minimize interference with the settling of interfering microspheres.

16. The method of claim 1, wherein the shape of the bottom of the microsphere collector is circular, tubular, funnel-shaped, or between shapes.

17. The method of claim 1 wherein the tube that draws the microspheres from the microsphere collector has an enlarged inlet.

18. The method of claim 1, comprising a container of the final formulation after post-treatment, which can be used to mix the desired additives.

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